The present invention relates to an automated system for the immunoassay of subnanogram quantities of certain compositions by molecular fluorescence.
The quantitative determination of small amounts of clinically significant compounds, such as metabolites, hormones, drugs and proteins are of recognized diagnostic importance. Radioimmunoassay (RIA) has become the standard method for making such determinations because of its sensitivity and specificity.
However, RIA has certain drawbacks. The radioactivity associated with RIA may present psychological or physical health hazards to the technologists, requires special licensing from nuclear regulatory agencies, requires the special disposal of wastes, limits the useful life of a reagent kit to a few months at the most, and requires relatively expensive instrumentation. To circumvent these drawbacks, alternative methods including fluorescence immunoassay (FIA) have been developed.
FIA is a technique in which a fluorescent molecule is substituted for the radioactive label used in RIA. Some of the advantages of FIA are: no radioactivity, a much longer useful lifetime of the test components or chemicals necessary for the assay, and relatively less expensive instrumentation for performing the assays.
By way of background, the commonly owned co-pending U.S. patent application bearing Ser. No. 875,475, filed Feb. 6, 1978 for SOLID PHASE IMMUNOFLUORESCENT ASSAY METHOD, now U.S. Pat. No. 4,201,763, describes in detail a FIA method for antigens (or haptens) and their antibodies through the use of an immune reactant related to the antibody or antigens to be determined which is covalently bonded or coupled to polymeric particles whose size permits direct measurement of a labelled immunological reagent's fluorescence in an aqueous suspension thereof by direct optical spectroscopy. A key to the method described in that U.S. patent application lies in the selection of certain types of polymeric particles in sizes which provide a substantially homogeneous suspension during execution of the assay. It has been discovered that such a condition exists and that direct fluorometric measurements can be made when the polymeric particles have a size of about 0.1-10 microns.
Utilizing such particles, an appropriate immune reactant immunologically related to unknown antigen or antibody to be determined is covalently bonded thereto. The particles, unknown immune reactant, and appropriate fluorescently labelled immune reactant are mixed under conditions so that a quantity of the labelled immune rectant proportional to the concentration of the unknown immune reactant is immunologically bound, directly or indirectly, to the particles.
In accordance with said co-pending U.S. patent application, the FIA provides water insoluble hydrophilic polymeric particles of about 0.1-10 microns in size and having covalently bonded thereto the immunological homolog for an antigen or antibody to be determined. The particles are combined with the antigen or the antibody to be determined in an aqueous solution to form an immunological bond therebetween. A fluorescently labelled antigen or antibody corresponding to the antigen or antibody to be determined is immunologically bound to the particles.
Any suitable water insoluble polymeric particle may be utilized in the FIA described in said U.S. patent application. Generally, the particle will be in spherical or bead form and will be selected from polymers which can be derivatized to give a terminal primary amine, terminal carboxyl, or hydroxide group. The antibody or antigen is then immobilized on the particle under conventional reaction conditions to produce a covalent bond therebetween. Useful polymeric particles are formed, for example from crosslinked polyacrylamides. Other suitable polymeric particles are described in said U.S. patent application and in the references cited therein.
The particles are then physically separated, usually by centrifuging them, typically at 1500 g to pack the particles at the bottom of the test tube into a pallet. The supernatant is decanted, to the extent necessary the tube or vial is blotted dry and a barbital buffer is added to the pellet in the test tube to reconstitute it and resuspend the particles to form a suspension which includes the fluorescent particles.
The suspension is then analyzed on a fluorometer to determine the concentration of fluorescent particles in the sample to obtain information from which unknown antigen or antibody can be determined.
As has been customary in the past, these tests have heretofore been performed manually one after the other. This required, inter alia, a vigorous manual shaking of the test tube to reconstitute each pellet and resuspend the fluorescent particles. To obtain an accurate test it is, of course, necessary that the suspension be uniform which prolonged the time during which the tube had to be shaken. Thereafter, the sample was fluorometrically analyzed, either in the test tube or by pouring it from the tube into a suitable container of a fluorometer.
This procedure is time-consuming and requires the constant close supervision by a highly skilled technician. More importantly, it gives no assurance that an adequate mixing of the sample has taken place. Without such mixing, however, the ultimate readout is inaccurate and can render the entire test of questionable value. Further, the test is relatively expensive because of the close and constant supervision it requires.
A key to the success of FIA is the reliability and accuracy of the fluorometer over extended periods of time. In this regard, prior art fluorometers had certain shortcomings which could affect the ultimate readout and thus compromise the accuracy of the test. Conventional fluorometers that operate in an analog mode are unsatisfactory because of the relative insensitivity of such fluorometers when measuring the low light intensities encountered when performing FIA.
Better accuracy can be attained with photon-counting fluorometers which are relatively simple and inexpensive to construct. Robert E. Curry et al discuss the construction of photon-counting fluorometers (hereinafter "fluorometer" unless otherwise indicated) in "Design and Evaluation of a Filter Fluorometer that Incorporates a Photon-Counting Detector" on pages 1259-1264 of Clinical Chemistry, Vol. 19, No. 11, 1973, although the use of such fluorometers in conjunction with FIA has not heretofore been considered. The Article notes that photo-counting is an effective method for minimizing dark current contributions in photomultiplier tubes since electrons emitted from the dynodes are amplified less than electrons emitted from the photocathode and level discriminating circuitry can be used to differentiate between the dark current and photon signals.
For the determination of small amounts (i.e. from subnanomolar levels up) of clinically significant compounds by FIA, accuracy problems are, of course, not fully solved by employing a photo-counting fluorometer. Stray light, a non-uniform suspension of the fluorescent beads, light scattering, a variation in the magnitude of the samples' own fluorescence as well as changes in the primary light intensity all adversely affect the ultimate readout and lessen its accuracy. In addition, existing FIA methods must rely on an essentially manual, sample by sample determination of the fluorescence which requires the constant attention of highly skilled and, therefore, costly operations. This in turn has a tendency to drive up the already high costs for such tests.